Gene and cell therapy using cell fusion technology

ABSTRACT

A gene and cell therapy using a cell fusion technology is proposed. Cells overexpressing hemagglutinin neuraminidase (HN) and fusion (F) proteins have effects of enhancing cell fusion with other cells, restoring cell damage through the cell fusion with damaged cells, and transferring a normal gene. Therefore, when a vector including genes encoding the HN and F proteins of the present invention or a cell transformed with the vector is clinically applied to neurodegenerative diseases, muscular diseases, and the like, an effect of reducing the damage of damaged cells through cell fusion can be expected.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/873,488 filed Jan. 17, 2018, which is a national stage application ofPCT/KR2016/008444 filed Aug. 1, 2016, which claims priority to KR10-2015-0101577, filed Jul. 17, 2015, the entire disclosure of which isincorporated herein by reference.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(8-PK0172383CON-SequenceListing.txt; Size: 4,059 bytes; and Date ofCreation: Mar. 27, 2018) is herein incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to gene and cell therapy using a cellfusion technology, and more particularly, to gene and cell therapeuticagents using a cell fusion technology capable of enhancing cell fusionwith other cells by transducing hemagglutinin neuraminidase (HN) andfusion (F) genes into cells and overexpressing the transduced cells andrestoring cell damage through cell fusion with damaged or dying cells orcells having gene abnormality.

Description of the Related Art

Generally, diseases and aging are progressed by cell damage andapoptosis. The common diseases that cause the cell damage and theapoptosis include neurodegenerative diseases, myopathy, and the like.

With the rapid increase in the elderly population, neurodegenerativediseases including damages of the brain, the spine and the peripheralnerves have been continuously increased. The causes of theneurodegenerative diseases are not clear yet. In addition, apathological mechanism of each neurodegenerative disease is known to acta little different mechanism. However, common causes include abnormalprotein aggregation, dysfunction of mitochondria, abnormality ofintracellular trafficking, oxygen radical injury, excitatory toxicity,autophagy/proteosomal dysfunction, neuroinflammation, deficiency ofneurotrophic factors, abnormality of RNA metabolism, and the like. Sincethese various pathological mechanisms act, it is difficult to treatdiseases by a therapeutic agent acting on any one mechanism, and thus,almost all clinical trials have so far failed. Therefore, a therapeuticagent that acts on a wider mechanism and has a powerful effect isrequired. These neurodegenerative diseases include Alzheimer's disease,Parkinson's disease, amyotrophic lateral sclerosis (ALS, Lou Gehrig'sdisease) which has a rapid progressing speed of the disease and the mostserious severity of the aftereffect, and the like. Particularly, theALS-related therapeutic agents are almost not existent except forriruzole and edaravone which are approved by the FDA in US and have onlyan effect of the prolonged survival for about 3 months or slightlyslowing deterioration of the physical function respectively, and thus,the development of a therapeutic technology is urgent. Otherneurodegenerative diseases also have no therapeutic agent for a completecure, and may be diseases in which a new therapeutic method including astem cell therapy is desperately required.

Currently, various therapeutic methods, such as cell transplantation andthe administration of drugs to improve the symptoms, are proposed totreat the neurodegenerative diseases, and especially recently, there isattention on cell therapy. However, a conventional cell therapytechnology aims to insert health cells (alternatively, stem cells) intoa diseased region to replace dead cells or improve an ambientenvironment of the dying cells to regenerate the dying cells, but theattempt has no effect or a slight effect in many preclinical or clinicaltrials. Further, in the case of neuronal cells, it is very important toform a neural circuit in terms of a function unlike other organs, andthus, it is very difficult for the cell supplied from the outside to bedifferentiated into the neuronal cells to restore the existing neuralcircuit as it is. Accordingly, in addition to the conventional methods,it is urgent to develop a new therapeutic method for reducing orprotecting neuronal cell damage.

Meanwhile, as diseases causing cell damage, Duchenn muscular dystrophy(DMD) and Backer muscular dystrophy (BMD) are included in musculardiseases, and these diseases are caused by abnormality of a dystrophingene existing in an X chromosome and about ⅓ thereof is caused bynatural mutation and the rest is caused by sex-linkage. Both the DMD andthe BMD are caused by the abnormality of the same gene, but the DMD iscalled a case in which a phenotype is severe due to frame-shift mutationand the like. In the case of the DMD, since the course of the disease ispoor, in 9 to 13 years of age, almost all patients are unable to walkand may be accompanied by cognitive decline as well as weakness ofmuscles accompanied by cardiomyopathy and respiratory distress to leadto death.

In the case of the DMD, recently, a method of attempting treatment byexon skipping has emerged. Since the exon skipping targets a splicingenhancer sequence of exon 51 of a dystrophin gene and has a principlethat restores only a reading frame converting the severe mutation to aless severe gene mutant, the exon skipping may not be a completetreatment alternative and is not a treatment method for targeting allDMDs.

Therefore, the present inventors made efforts to develop a therapeuticagent for cell damage-related diseases including neurodegenerativediseases, muscular diseases, and the like, and as a result, found thatcells overexpressing hemagglutinin neuraminidase (HN) and fusion (F)proteins have enhanced cell fusion with other cells by the HN and Fproteins and high ability of restoring cell damage in the dying cells,and normal dystrophin was expressed by cell fusion. In addition, thepresent inventors found that the present invention can be usefully usedto restore the cell damage in diseases causing the cell damage such asneurodegenerative diseases and muscular diseases and introduce a normalgene, and then completed the present invention.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method for reducingcell damage including: administering a vector including genes encodinghemagglutinin neuraminidase (HN) and fusion (F) proteins or a celltransformed with the vector to a subject with a therapeuticallyeffective dose.

Another object of the present invention is to provide a therapeuticagent and a therapeutic method for cell damage-related diseasesincluding neurodegenerative diseases, muscular diseases, and the likeusing hemagglutinin neuraminidase (HN) and fusion (F) genes.

An exemplary embodiment of the present invention provides a method forreducing cell damage including: administering a vector including genesencoding hemagglutinin neuraminidase (HN) and fusion (F) proteins or acell transformed with the vector to a subject with a therapeuticallyeffective dose.

Another exemplary embodiment of the present invention provides a methodfor treating neurodegenerative diseases or neurological diseasesincluding: administering a vector including genes encoding hemagglutininneuraminidase (HN) and fusion (F) proteins or a cell transformed withthe vector to a subject with a therapeutically effective dose.

Yet another exemplary embodiment of the present invention provides amethod for treating degenerative muscular diseases or muscular diseasesincluding: administering a vector including genes encoding hemagglutininneuraminidase (HN) and fusion (F) proteins or a cell transformed withthe vector to a subject with a therapeutically effective dose.

According to the present invention, since the cells overexpressing thehemagglutinin neuraminidase (HN) and fusion (F) proteins have excellentcell fusion ability with other cells by the HN and F proteins andability of restoring cell damage in dying cells and transferring normalgenes, the present invention can be used to reduce cell damage andintroduce normal genes in diseases having cell damage and geneabnormality, such as neurodegenerative diseases and muscular diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other aspects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 illustrates an effect of inhibiting cell death of a dying NSC34motorneuron cell line by cell fusion with hATMSCs according to anexemplary embodiment of the present invention;

FIG. 2 illustrates an expression analysis of Bax and Bcl-xL in a NSC34cells, hATMSCs, apoptotic NSC34 cells and fused cells (apoptotic NSC-34cells with hATMSCs) according to the exemplary embodiment of the presentinvention;

FIG. 3 is a schematic diagram for preparing expression vectors insertedwith HN and F according to the exemplary embodiment of the presentinvention;

FIG. 4 illustrates an analysis of expression of HN and F mRNAs inHN/F-hATMSCs according to the exemplary embodiment of the presentinvention;

FIG. 5 illustrates an imaging analysis of expression of HN and Fproteins in HN/F-hATMSCs according to the exemplary embodiment of thepresent invention;

FIG. 6 illustrates an immunophenotyping analysis of HN/F-hATMSCsaccording to the exemplary embodiment of the present invention;

FIG. 7 illustrates an analysis of cell fusion ability with a NSC34motorneuron cell line in HN/F-hATMSCs according to the exemplaryembodiment of the present invention;

FIG. 8 illustrates an analysis of expression of ChAT and CD105 in fusedcells of the NSC34 motorneuron cell line and the HN/F-hATMSCs accordingto the exemplary embodiment of the present invention;

FIG. 9 illustrates a proteomic analysis result among the NSC34motorneuron cell line, the dying NSC34 motorneuron cell line, and thefused cell line (dying NSC 34 with the HN/F-hATMSCs) according to theexemplary embodiment of the present invention;

FIG. 10 illustrates a heatmap analysis result among the NSC34motorneuron cell line, the dying NSC34 motorneuron cell line, and thefused cell line (dying NSC 34 with the HN/F-hATMSCs) according to theexemplary embodiment of the present invention;

FIG. 11 illustrates an analysis of expression of genes DDB1, HMGB1, andMSH2 mRNAs related with cell repair in the NSC34 motorneuron cell line,the dying NSC34 motorneuron cell line, and the fused cell line (dyingNSC 34 with the HN/F-hATMSCs) according to the exemplary embodiment ofthe present invention;

FIG. 12 illustrates an analysis of expression of HN and F mRNAs inHN/F-heLa according to an exemplary embodiment of the present invention;

FIG. 13 illustrates an analysis of cell fusion ability with a N2Aneuroblastoma cell line in HN/F-HeLa according to the exemplaryembodiment of the present invention;

FIG. 14 illustrates confirming a TDP-43 binding motif which is atranscriptional factor (TF) in a promoter region of a gene DDB1 relatedwith cell repair selected according to the exemplary embodiment of thepresent invention;

FIG. 15 illustrates a mouse DDB1 (mDDB1) promoter having TDP-43 bindingsequences and a schematic diagram for preparing mDDB1 specific primersaccording to the exemplary embodiment of the present invention;

FIG. 16 illustrates an analysis of mDDB1 expression by TDP-43 in fusedcells of a HeLa cell line transduced with GFP-TDP-43 and the N2Aneuroblastoma cell line according to the exemplary embodiment of thepresent invention;

FIG. 17 illustrates an imaging analysis of translocation of TDP-43 froma nucleus of HeLa into a nucleus of the N2A neuroblastoma cell line inthe fused cells of the HeLa cell line transduced with GFP-TDP-43 and theN2A neuroblastoma cell line according to the exemplary embodiment of thepresent invention;

FIG. 18 illustrates an intra-spinal cord injection method for injectingHN/F-hATMSCs in a cell damage-related mouse model (G93A SOD1 Tg mice)according to an exemplary embodiment of the present invention;

FIG. 19 illustrates an analysis of motion performance in a G93A SOD1 Tgmouse injected with HN/F-hATMSCs according to the exemplary embodimentof the present invention;

FIG. 20 illustrates survival rates and dystrophin expression ofHN/F-hATMSCs in a skeletal muscle tissue of an mdx mouse injected withHN/F-hATMSCs according to the exemplary embodiment of the presentinvention; and

FIG. 21 is a schematic diagram illustrating a cell damage restorationmechanism by HN/F protein overexpressed cells in a cell damage-relateddisease.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Hereinafter, the present invention will be described in detail by thefollowing Examples.

The present invention provides a vector including genes encodinghemagglutinin neuraminidase (HN) and fusion (F) proteins.

In the present invention, the HN and F proteins may be derived fromsendai virus, human immunodeficiency virus 1, influenza virus orvesicular stomatitis virus, but is not limited thereto. Various types ofHN and F proteins having various origins and/or full lengths and/orfragments may be used as long as these proteins may promote the fusionof cells, inhibit the death, and restore the damage to the damaged cellsthrough the same or similar mechanism as or to these proteins (Eckert DM, Kim P S. Annu Rev Biochem. 2001; 70:777-810; Sapir A, Avinoam O,Podbilewicz B, Chernomordik L V. Dev Cell. 2008 January; 14(1):11-21;Kouris N A, Schaefer J A, Hatta M, Freeman B T, Kamp T J, Kawaoka Y,Ogle B M. Stem Cells Int. 2012; 2012:414038).

The hemagglutinin neuraminidase (HN) according to the present inventionis a glucoprotein expressed in viruses and may exist as a single virusprotein having activities of both hemagglutinin and neuraminidase orseparate proteins, but is not limited thereto and all of the proteinsmay be included in the present invention as long as the proteins achievean effect according to the present invention. Examples of the former mayinclude those derived from paramyxoviruses such as a mumps virus, asendai virus, a human parainfluenza virus, or an avian Newcastle diseasevirus and examples of the latter may include those derived from aninfluenza virus. These proteins and nucleic acid sequences are known,and for example, the hemagglutinin neuraminidase may refer toinformation in Enzyme entry (http://enzyme.expasy.org) EC:3.2.1.18 andthe protein sequences are derived from, for example, sendai viruses andpublished in GenBank accession no. AAB06288.1. In one embodimentaccording to the present invention, a sendai virus-derived HN singleprotein which may be prepared according to a method in Example 2 of thepresent invention is used.

Further, the F protein is called a glucoprotein which is used inintercellular fusion or fusion/entry of virus into cells, endocytosis,and membrane trafficking. In one embodiment according to the presentinvention, virus-derived F proteins are used and divided into classes I,II, and III according to structural features. Examples of the class Iinclude GP2 of ebola virus, Mo-55 of moloney murine leukemia virus(MoMuLv), gp41 of immunodeficiency virus HIV, simian virus (SIV) gp41,and the like. Examples of the class II include SFV E1, TBEV E, and thelike. Examples of the class III include glycoprotein B (gB) of herpessimplex virus (HSV), gB of epstein-barr virus (EBV), protein G ofvesicular stomatitis virus (VSV), glycoprotein gp64 of beculovirus, andthe like. Such fusion glycoproteins and nucleic acid sequences arepublished and for example, published in GenBank Accession no.AAC82300.1.

In the present invention, the genes encoding the HN and F proteins maybe used as full-length and/or fragmental types. Particularly, wide-typegene sequences encoding the proteins disclosed in the present inventionand fragments thereof include genes in which some of base sequences areartificially modified so that features such as expression in cells orstability of the proteins are advantageous, and naturally found genes inwhich some of base sequences are modified, or all fragments thereof. Themodification of the gene base sequences may include or not modificationsof corresponding amino acids, and the modifications of the amino acidsinclude mutants, derivatives, alleles, variants, and homologues, whichencode proteins consisting of amino acid sequences in which one or moreamino acids are substituted, deleted, added and/or inserted in a proteinencoded by the gene causing the modification. When the mutation of thegene base sequences does not include the modification of the amino acidsin the protein, the mutation includes, for example, degeneracy mutation,and these degeneracy mutants are included in the gene of the presentinvention.

The artificial modification of the gene base sequences may be preparedby methods known to those skilled in the art, for example, site-directedmutagenesis (Kramer et al, 1987), error-induced PCR (Cadwell, R. C. andG. F. Joyce. 1992. PCR methods Appl., 2:28-33), point mutation (Sambrookand Russel, Molecular Cloning: A Laboratory Manual, 3rd Ed. 2001, ColdSpring Harbor Laboratory Press), and the like.

In the present invention, the vector means a means for expressing atarget gene in host cells. The vector includes elements for expressingthe target gene and may include a replication origin, a promoter, anoperator gene, a transcription terminator, and the like and may furtherinclude a suitable enzyme site (for example, a restrictive enzyme site)for introduction into a genome in the host cells and/or a selectionmarker for confirming successful introduction into the host cells,and/or a ribosome binding site (RBS) for translation to the protein, aninternal ribosome entry site (IRES), and the like. The vector mayfurther include a transcription regulator (for example, an enhancer andthe like) in addition to the promoter.

Further, the vector may be a plasmid DNA, a recombinant vector, or othervectors, which are known in the art, and particularly, may be a linearDNA plasmid DNA, a non-viral recombinant vector, a viral recombinantvector, or an inducible gene expression vector system, and the viralrecombinant vector may be retroviruses, adenoviruses, adeno-associatedviruses, helper-dependent adenoviruses, herpes simplex viruses,lentivirus vectors or vaccinia viruses, but is not limited thereto.

Further, the vector means an expression vector for gene therapy. Theterm “gene therapy” refers to a method of inserting a normal gene intocells having gene abnormality or proving a new function to normalize itsfunction in order to treat various genetic diseases caused by the geneabnormality. Accordingly, in the present invention, the expressionvector for gene therapy means an expression vector which may provide anew function through cell fusion between cells having gene abnormalityand normal cells by transferring hemagglutinin neuraminidase (HN)/fusion(F) protein genes into normal cells in the body and therefore normalizeits function.

In one embodiment of the present invention, the HN/F protein genes maybe introduced into the cells according to the present invention bypreparing primers which can specifically recognize the genes from knownsequences, amplifying the genes through polymerase chain reaction usingthe primers, and introducing the amplified genes into the expressionvector, as described below. The introduction method is known and forexample, may include liposome-mediated transduction, calcium phosphatetransduction, DEAE-dextran mediated transduction, positively chargedlipid mediated transduction, electroporation, transduction using a phagesystem, infection using a virus, or the like, but the present inventionis not limited thereto.

Further, the present invention provides a cell transformed with a vectorincluding genes encoding hemagglutinin neuraminidase (HN) and fusion (F)proteins.

In the present invention, the term “transformation” means a phenomenonin which a genetic property of a living organism is changed by DNA givenfrom the outside, that is, a genetic trait is changed while DNA isintroduced to the cells thereof when DNA, a kind of nucleic acidextracted from cells in any system of the living organism is introducedinto living cells in another system.

In the present invention, the transformed cells may be stem cells,progenitor cells, or animal cells, but the present invention is notlimited thereto. Further, the stem cells may be particularly embryonicstem cells, adult stem cells, and induced pluripotent stem cells (iPS)and more particularly adult stem cells (mesenchymal stem cells), but thepresent invention is not limited thereto. Further, the cells may beautologous or allogeneic, or allogenic or xenogenic cells. Mostpreferably, since the cells are autologous-derived and derived from arecipient, there is no problem in immune response when a pharmaceuticalcomposition is administered and there is an advantage in safety.

The adult stem cells refer to undifferentiated stem cells found in thewhole adult even after an embryonic development stage. The adult stemcells have a site-specific differentiation in which the cells themselvesare differentiated according to characteristics of surrounding tissues.The adult stem cells may be derived from various adult cells, such asbone marrow, blood, brain, skin, fat, skeletal muscle, umbilical cord,cord blood, and the like. Specific examples thereof may includemesenchymal stem cells (MSC), skeletal muscle stem cells, hematopoieticstem cells, neuronal stem cells, hepatic stem cells, adipose-derivedstem cells, adipose-derived progenitor cells, vascular endothelialprogenitor cells, and the like, but the present invention is not limitedthereto.

In addition, the mesenchymal stem cells are adult stem cells obtainedfrom the respective parts of the body that have already become adult,and refer to pluripotent or multipotent cells capable of differentiatinginto various cells, for example, adipocytes, motorneuron cells, and thelike, as stem cells isolated from cord blood, fat, bone marrow, blood,dermis or periosteum. Further, the mesenchymal stem cells may beeffectively engrafted from allogenic or xenogenic recipients withoutusing immune inhibitors. The mesenchymal stem cells may be animal,particularly mammal, and more particularly human mesenchymal stem cells.

In one embodiment of the present invention, the mesenchymal stem cellsare stem cells derived from adipose tissue. The mesenchymal stem cellsderived from the adipose tissue have a practical advantage of being ableto receive a large amount unlike bone marrow, amniotic fluid, and cordblood stem cells, about 1% of the adipocytes has been estimated as thestem cells, and recently, the adipose-derived stem cells are highlyuseful due to infinite supplyability when considering that a cosmeticsurgery widely performed in the advanced countries is liposuction.

A process of obtaining the mesenchymal stem cells will be described asfollows. The mesenchymal stem cells are isolated from mammals includinghumans or mice, preferably human mesenchymal stem cell sources, forexample, adipose tissue, blood or bone marrow. Next, the cells arecultured in a suitable medium. In the culture process, the floatingcells are removed and the cells attached to a culture plate aresubcultured to finally obtain established mesenchymal stem cells.

In addition, a process of isolating and culturing a very small amount ofmesenchymal stem cells in bone marrow and the like, is well known in theart, and for example, there is disclosed in U.S. Pat. No. 5,486,359.

As the medium to be used in the above process, any medium generally usedfor culturing the stem cells may be used. Preferably, the medium is amedium containing serum (for example, fetal bovine serum, horse serumand human serum). The medium to be used in the present inventionincludes, for example, RPMI series, Eagles's MEM (Eagle's minimumessential medium, Eagle, H. Science 130:432(1959)), α-MEM (Stanner, C.P. et al., Nat. New Biol. 230:52(1971)), Iscove's MEM (Iscove, N. etal., J. Exp. Med. 147:923(1978)), 199 media (Morgan et al., Proc. Soc.Exp. Bio. Med., 73:1(1950)), CMRL 1066, RPMI 1640 (Moore et al., J.Amer. Med. Assoc. 199:519(1967)), F12 (Ham, Proc. Natl. Acad. Sci. USA53:288(1965)), F10 (Ham, R. G. Exp. Cell Res. 29:515(1963)), DMEM(Dulbecco's Modified Eagle's medium, Dulbecco, R. et al., VirProcy8:396(1959)), a mixture of DMEM and F12 (Barnes, D. et al., Anal.Biochem. 102:255(1980)), Way-mo, h's MB752/1 (Waymo, h, C. J. Natl.Cancer Inst. 22:1003(1959)), McCoy's 5A (McCoy, T. A., et al., Proc.Soc. Exp. Biol. Med. 100:115(1959)) and MCDB series (Ham, R. G. et al.,In Vitro 14:11(1978)), but the present invention is not limited thereto.In the media, other ingredients, for example, antibiotics, or antifungalagents (e.g., penicillin, streptomycin), glutamine, and the like may beincluded. A general description of the media and culture is disclosed inR. Ian Freshney, Culture of Animal Cells, Alan R. Liss, Inc., New York(1984), which is incorporated in this specification by reference.

The mesenchymal stem cells may be confirmed by, for example, a flowcytometry. The flow cytometry is performed by using a specific surfacemarker of the mesenchymal stem cells. In one embodiment of the presentinvention, the mesenchymal stem cells according to the present inventionhave CD29, CD44 and CD90 as markers and phenotypes of CD34, CD45 andHLA-DR as negative markers.

In addition, the inducible pluripotent stem cells can be prepared tomultipotent stem cells such as embryonic stem cells by introducing fourspecific genes that induce dedifferentiation into somatic cells such asskin cells of a non-pluripotent adult, and then expressing the cells orextracting a dedifferentiation inducible protein prepared from the cellsintroduced with the four genes to inject the extracted protein intosomatic cells again, which are called inducible multipotent stem cellsor dedifferentiation stem cells. The inducible pluripotent stem cellsare used for stem cell therapy using the inducible pluripotent stemcells, cell-based studies in disease model and drug development byproducing inducible pluripotent stem cells and differentiating the stemcells in vitro by obtaining somatic cells from patients to studyprogress of various diseases, and the like.

Further, the progenitor cell is a cell at a stage before the shape andfunction of a specific cell are established, and is a cell that can bedifferentiated into cells of a specific cell line or can be formed intoa specific type of tissue, and means a cell having self-renewal, but anextremely limited differentiation. Endoderm progenitor cells, mesodermalprogenitor cells, and ectoderm precursor cells are all included therein.

Further, the animal cell is a functional and structural basic unitoriginating from an animal including a human and may be included in thescope of the present invention if it is a cell originating from ananimal including a human being (for example, a mammal such as a monkey,a dog, a goat, a pig, a mouse, and the like). Accordingly, the animalcells of the present invention are not limited thereto, but includeepithelial cells, endothelial cells, muscular cells, germ cells, skincells (e.g., fibroblasts, keratinocytes), immune cells, cancer cells andthe like. As specific examples, chinese hamster ovary (CHO) cells, mousemyeloma (NSO) cells, baby hamster kidney (BHK) cells, Sp2/0 (mousemyeloma) cells, human retinal cells, HUVEC cells, HMVEC cells, COS-1cells, COS-7 cells, HeLa cells, HEK-293 cells, HepG-2 cells, HL-60cells, IM-9 cells, Jurkat cells, MCF-7 cells or T98G cells may beexemplified, but the present invention is not limited thereto.

Further, the present invention provides a pharmaceutical composition fortreating cell damage-related diseases including a vector including genesencoding hemagglutinin neuraminidase (HN) and fusion (F) proteins or acell transformed with the vector.

In the present invention, the vector or the cell transformed with thevector are the same as the description of the vector and the cells, andthe detailed description thereof refers to the above contents.Hereinafter, only a specific configuration of the pharmaceuticalcomposition for treating cell damage-related diseases will be described.

In the present invention, the cell damage may be neuronal cell damage ormuscular cell damage, and the neuronal cell damage may be caused byneurodegenerative diseases, neurological diseases, degenerative braindiseases, spinal cord injury, peripheral nerve injury, or neuronal celldeath and the muscular cell damage may be caused by degenerativemuscular disease, muscular diseases, and muscular cell damage due togene anomalies, but the present invention is not limited thereto.

In the present invention, the cell damage-related diseases may includeneurodegenerative diseases, neurological diseases, degenerative musculardiseases, or muscular diseases, but the present invention is not limitedthereto.

The neurodegenerative disease or the neurological disease may includeAlzheimer's disease (AD), dementia, multi-infarct dementia (MID),frontotemporal dementia, dementia with Lewy bodies, mild cognitiveimpairment, corticobasal degeneration, Parkinson's disease (PD),multiple system atrophy (MSA), Huntington's disease, spinal muscularatrophy, spinal bulbar muscular atrophy, progressive supranuclear palsy(PSP), metabolic brain disease, depression, epilepsy,dentatorubropallidoluysian atrophy (DRPLA), spinocerebellar ataxia,amyotrophic lateral sclerosis (ALS), multiple sclerosis, primary lateralsclerosis, progressive bulbar palsy, glaucoma, stroke, brain ischemia,post-encephalitic parkinsonism, Tourette's syndrome, restless legssyndrome, or attention deficit disorders with hyperactivity, but thepresent invention is not limited thereto.

The degenerative muscular disease or the muscular disease may includemyopathy, congenital myopathy, congenital muscular dystrophy, Duchennemuscular dystrophy, Becker muscular dystrophy, Limb Girdle musculardystrophy, facioscapulohumeral muscular dystrophy, oculopharyngealmuscular atrophy, distal muscular dystrophy, Emery-Dreifuss musculardystrophy, myotonic dystrophy, Barth syndrome, heart failure, orX-linked dilated cardiomyopathy, but the present invention is notlimited thereto.

In the present invention, since it is confirmed that the cellsoverexpressing the HN/F proteins promote cell fusion with other cells bythe HN/F proteins, a normal gene is transferred and expressed throughfusion with the damaged cells, and the expression of the gene forrestoring the cell damage is regulated, the vector including the genesencoding the HN/F proteins or the cell transformed with the vector maybe usefully used to reduce and/or restore the cell damage in diseasescausing cell damage and gene abnormality, such as neurodegenerativediseases or degenerative muscular diseases.

The pharmaceutical composition according to the present invention may beformulated in a suitable form together with a pharmaceuticallyacceptable carrier which is generally used. The pharmaceuticallyacceptable carrier may include, for example, water, suitable oils,saline, parenteral administration carries such as aqueous glucose andglycol, and the like, and further include stabilizers and preservatives.The suitable stabilizer includes an antioxidant such as sodium hydrogensulfite, sodium sulfite, or ascorbic acid. The suitable preservativeincludes benzalcohol chloride, methyl- or propyl-paraben, andchlorobutanol. In addition, the composition for cell therapy accordingto the present invention may suitably include suspending agents,solubilizers, stabilizers, isotonizing agents, preservatives, adsorptioninhibitors, surfactants, diluents, excipients, pH adjusters, analgesicagents, buffers, antioxidants, and the like according to theadministration method or the formulation, if necessary. Thepharmaceutically acceptable carriers and formulations suitable for thepresent invention, including those exemplified above, are described indetail in the document [Remington's Pharmaceutical Sciences, latestedition].

The pharmaceutical composition of the present invention is formulated byusing a pharmacologically acceptable carrier and/or excipient accordingto a method that may be easily performed by those skilled in the art tobe prepared in a unit dosage form or prepared by intrusion into amulti-dose container.

Further, the pharmaceutical composition may also be administered by anydevice in which active ingredients may move to the target cell. Thepharmaceutical composition of the present invention may be included witha therapeutically effective dose for treatment of diseases. In thepresent invention, the term “treatment’ means reversing or mitigatingone or more symptoms of a disease or a disorder to which the term isapplied, or inhibiting or preventing the progress thereof, unlessotherwise stated. The term “therapeutically effective dose” means anamount of an active ingredient or a pharmaceutical composition whichinduces a biological or medical response in tissue systems, animals, orhumans which is considered by researchers, veterinarian, physician, orother clinicians, and includes an amount of inducing mitigation ofsymptoms of diseases or disorders to be treated. It is apparent to thoseskilled in the art that the active ingredients included in thepharmaceutical composition of the present invention are changedaccording to an effect.

Therefore, the optimal content of the pharmaceutical composition may beeasily determined by those skilled in the art, and may be adjustedaccording to various factors including a type of disease, severity ofthe disease, contents of other ingredients contained in the composition,a type of formulation, and an age, a weight, a general health condition,a gender, and a diet of a patient, an administration time, aadministration route, a secretion ratio of the composition, a treatmentperiod, and simultaneously used drugs.

In one embodiment of the present invention, the pharmaceuticalcomposition is administered intravenously or intrathecally.

It is important to include an amount capable of obtaining a maximumeffect by a minimum amount without side effects by considering all ofthe factors. For example, the dose of the composition of the presentinvention may be 0.1×10⁵ to 1.0×10⁸ cells/kg (body weight) and morepreferably 0.5×10⁶ to 1.0×10⁷ cells/kg (body weight) of a celltransformed with the vector including the genes encoding the HN and Fproteins as active ingredients. However, the dose thereof may bevariously prescribed by factors such as a formulation method, anadministration type, age, weight, and gender of a patient, apathological condition, food, an administration time, an administrationroute, an excretion rate, and response susceptibility, and those skilledin the art may suitably adjust the dose by considering the factors. Thenumber of administrations may be one or two times within a range ofclinically acceptable side effects, and even in administration sites,the composition may also be administered to one site or two or moresites. Even in the animals other than the human, the same dose as thatof the human per kg may be administered, or an amount obtained byconverting the dose by, for example, a volume ratio (for example, anaverage value) in ischemic organs (heart and the like) between targetanimal and human and the like may be administered. Examples of theanimal to be treated according to the present invention may includehumans, and mammals for other purposes, and particularly, includehumans, monkeys, mice, rats, rabbits, sheep, cows, dogs, goats, horses,pigs, and the like.

Further, the present invention provides a method for reducing celldamage including: administering a vector including genes encodinghemagglutinin neuraminidase (HN) and fusion (F) proteins or a celltransformed with the vector to a subject, with a therapeuticallyeffective dose.

In the present invention, the vector or the cell transformed with thevector, the administration method and the dose thereof, and the like aredescribed the same as those of the vector, the cells, and theadministration method and the dose of the pharmaceutical compositionincluding the same, and thus, the detailed description will be based onthe contents.

In the present invention, the cell damage may be neuronal cell damage ormuscular cell damage, and the neuronal cell damage may be caused byneurodegenerative diseases, neurological diseases, degenerative braindiseases, spinal cord injury, peripheral nerve injury, or neuronal celldeath and the muscular cell damage may be caused by degenerativemuscular diseases, muscular diseases, and muscular cell damage due togene anomalies, but the present invention is not limited thereto.

In the present invention, the subject is a subject that suffers from theneurodegenerative diseases, the neurological diseases, the degenerativemuscular diseases, or the muscular diseases, and the more particularly,includes mammals, for example, humans, monkeys, mice, rats, rabbits,sheep, cows, dogs, goats, horses, pigs, and the like, which areadministered with the vector including the genes encoding the HN and Fproteins or the cell transformed with the vector to mitigate and/orrestore the cell damage caused by the diseases, for example, theneuronal cell damage or the muscular cell damage.

The neurodegenerative disease or the neurological disease may includeAlzheimer's disease (AD), dementia, multi-infarct dementia (MID),frontotemporal dementia, dementia with Lewy bodies, mild cognitiveimpairment, corticobasal degeneration, Parkinson's disease (PD),multiple system atrophy (MSA), Huntington's disease, spinal muscularatrophy, spinal bulbar muscular atrophy, progressive supranuclear palsy(PSP), metabolic brain disease, depression, epilepsy,dentatorubropallidoluysian atrophy (DRPLA), spinocerebellar ataxia,amyotrophic lateral sclerosis (ALS), multiple sclerosis, primary lateralsclerosis, progressive bulbar palsy, glaucoma, stroke, brain ischemia,post-encephalitic parkinsonism, Tourette's syndrome, restless legssyndrome, or attention deficit disorders with hyperactivity, but thepresent invention is not limited thereto.

In addition, the degenerative muscular disease or the muscular diseasemay include myopathy, congenital myopathy, congenital musculardystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, LimbGirdle muscular dystrophy, facioscapulohumeral muscular dystrophy,oculopharyngeal muscular atrophy, distal muscular dystrophy,Emery-Dreifuss muscular dystrophy, myotonic dystrophy, Barth syndrome,heart failure, or X-linked dilated cardiomyopathy, but the presentinvention is not limited thereto.

In the present invention, since it is confirmed that the cellsoverexpressing the HN/F proteins promote cell fusion with other cells bythe HN/F proteins, a normal gene is transferred and expressed throughfusion with the damaged cells, and the expression of the gene restoringthe cell damage is regulated, the vector including the genes encodingthe HN/F proteins or the cell transformed with the vector may beusefully used to reduce/or restore cell damage caused byneurodegenerative diseases, neurological diseases, degenerative braindiseases, spinal cord injury, peripheral nerve injury, neuronal celldeath, degenerative muscular diseases, muscular diseases, muscular celldamage due to gene abnormality, or the like.

Further, the present invention provides a method for treatingneurodegenerative diseases or neurological diseases including:administering a vector including genes encoding hemagglutininneuraminidase (HN) and fusion (F) proteins or a cell transformed withthe vector to a subject, with a therapeutically effective dose.

In the present invention, the vector or the cell transformed with thevector, the administration method and the dose thereof, and the like aredescribed the same as those of the vector, the cells, and theadministration method and the dose of the pharmaceutical compositionincluding the same, and thus the detailed description will be based onthe contents.

In the present invention, the neurodegenerative disease or theneurological disease may include Alzheimer's disease (AD), dementia,multi-infarct dementia (MID), frontotemporal dementia, dementia withLewy bodies, mild cognitive impairment, corticobasal degeneration,Parkinson's disease (PD), multiple system atrophy (MSA), Huntington'sdisease, spinal muscular atrophy, spinal bulbar muscular atrophy,progressive supranuclear palsy (PSP), metabolic brain disease,depression, epilepsy, dentatorubropallidoluysian atrophy (DRPLA),spinocerebellar ataxia, amyotrophic lateral sclerosis (ALS), multiplesclerosis, primary lateral sclerosis, progressive bulbar palsy,glaucoma, stroke, brain ischemia, post-encephalitic parkinsonism,Tourette's syndrome, restless legs syndrome, or attention deficitdisorders with hyperactivity, but the present invention is not limitedthereto.

In the present invention, since it is confirmed that the cellsoverexpressing the HN/F proteins promote cell fusion with other cells bythe HN/F proteins, a normal gene is transferred and expressed throughfusion with the damaged cells, and the expression of the gene restoringthe cell damage is regulated, the vector including the genes encodingthe HN/F proteins or the cell transformed with the vector may beusefully used to treat neurodegenerative diseases or neurologicaldiseases.

Further, the present invention provides a method for treatingdegenerative muscular diseases or muscular diseases including:administering a vector including genes encoding hemagglutininneuraminidase (HN) and fusion (F) proteins or a cell transformed withthe vector to a subject, with a therapeutically effective dose.

In the present invention, the vector or the cell transformed with thevector, the administration method and the dose thereof, and the like aredescribed the same as those of the vector, the cells, and theadministration method and the dose of the pharmaceutical compositionincluding the same, and thus the detailed description will be based onthe contents.

The degenerative muscular disease or the muscular disease may includemyopathy, congenital muscular dystrophy, Duchenne muscular dystrophy,Becker muscular dystrophy, Limb Girdle muscular dystrophy,facioscapulohumeral muscular dystrophy, oculopharyngeal muscularatrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy,myotonic dystrophy, Barth syndrome, heart failure, or X-linked dilatedcardiomyopathy, but the present invention is not limited thereto.

In the present invention, since it is confirmed that the cellsoverexpressing the HN/F proteins promote cell fusion with other cells bythe HN/F proteins, a normal gene is transferred and expressed throughfusion with the damaged cells, and the expression of the gene restoringthe cell damage is regulated, the vector including the genes encodingthe HN/F proteins or the cell transformed with the vector may beusefully used to treat degenerative muscular diseases or the musculardiseases.

Hereinafter, the present invention will be described in detail by thefollowing Examples. However, the following Examples just exemplify thepresent invention, and the contents of the present invention are notlimited to the following Examples.

EXAMPLE

Cell Culture

Human adipose tissue-derived mesenchymal stem cells (hATMSCs) obtainedby agreement and understanding of the K-STMECELL institutional reviewboard (IRB) were cultured in a RKCM culture medium (added with 10% FBSand provided from the K-STEMCELL) added with antibiotics. A mousemotorneuron cell line (NSC34 Motor Neuron-Like Hybrid Cell line,CEDARLANE, USA) was cultured in a Dulbecco's Modified Eagle's medium(DMEM) (added with 10% FBS) added with antibiotics. A mouseneuroblastoma cell line (N2A cell line, ATCC) was cultured in an Eagle'sMinimum Essential medium (EMEM) (added with 10% FBS) added withantibiotics. A human cervical cancer cell line (HeLa cell line, ATCC)was cultured in an Eagle's Minimum Essential medium (EMEM) (added with10% FBS) added with antibiotics. All of the cells were cultured at 37°C. under 5% CO₂ supply.

Example 1. Analysis of Apoptosis Inhibition Effect in Motorneuron CellLine by Cell Fusion Between Dying Motorneuron Cell Line andAdipose-Derived Mesenchymal Stem Cells

Motorneuron cell line NSC34 cells marked with a cell tracker CM-DiI(Thermofisher) were treated with 2.5 μM of thapsigargin as an apoptosisinducer and then cultured for 24 hours. After Annexin V staining,annexin V-positive NSC34 cells were isolated using an annexin V antibodyby a flow cytometry. hATMSCs and annexin V-positive NSC34 cells markedwith a green dye (Cell-Stalker™, Biterials) were fused using a Sendaivirus (HVJ) envelope cell fusion kit (GenomONE™-CF EX Envelope CellFusion Kit, COSMO BIO, Japan) according to a manufacturer's method, andthen V-positive cells were analyzed using a flow cytometry. As shown inFIG. 1 , 99.5% of annexin V-positive cells were found in a group ofNSC34 cells which were not fused with the hATMSCs, and 32.9% of annexinV-positive cells were found in a group of NSC34 cells which are fusedwith the hATMSCs. Such a result indicates that the death of the NSC34motoneuron cell line may be inhibited by the cell fusion with thehATMSCs.

Further, the apoptosis inhibition effect was confirmed at a molecularlevel. In summary, the NSC34 cell group which was fused with the hATMSCswas isolated through the flow cytometry and then expression of apro-apoptotic gene Bax and an anti-apoptotic gene Bcl-xL was analyzed byreverse transcription polymerase chain reaction (RT-PCR) andquantitative RT-PCR. As shown in FIG. 2 , as compared with the NSC34cell group which was not fused with the hATMSCs, in the NSC34 cell groupwhich was fused with the hATMSCs, the expression of Bax mRNA wassignificantly decreased and the expression of Bcl-xL mRNA wassignificantly increased.

Such a result indicates that apoptosis is inhibited through the decreasein expression of the pro-apoptotic gene Bax and the increase inexpression of the anti-apoptotic gene Bcl-xL when the dying NSC34motorneuron cell line was fused with the hATMSCs.

Example 2. Preparation of Human Adipose-Derived Mesenchymal Stem Cells(HN/F-hATMSCs) Overexpressing Hemagglutinin Neuraminidase (HN)/Fusion(F) Proteins

Genes of hemagglutinin neuraminidase (HN) and F protein (F) wereamplified by PCR using a Sendai virus genome as a template and a primershown in Table 1. The amplified DNAs were inserted into a pcDNA3.1expression vector, respectively, and cloned in E. coli stain DH5a toconfirm a sequence (the result was not shown). The cloned HN and Fproteins are the same as published sequences GenBank Accesssion No.AAB06288.1 and AAC82300.1, respectively.

TABLE 1 Primer sequence used in cloning of HN and F genes used in the present invention Target Product gene Primer sequencessize (bp) HN FP 5′-AAGCTTATGGAACAAAAACTCAT 1731CTCAGAAGAGGATCTGGATGGTGATA GGGGCAAACGTGACTCGTACTGG-3′ HN RP5′-GAATTCTCATCTTTTCTCAGCCA TTGCATCAAACCCACC-3′ F FP5′-AAGCTTATGCATCATCATCATCA 1704 TCATACAGCATATATCCAGAGATCAC AGTGCATCTC-3′F RP 5′-GAATTCTCATCTTTTCTCAGCCA TTGCATCAAACCCACC-3′ Partial5′-CGATCTCTGGATGTGTTAG-3′  411 HN FP Partial 5′-CCACACTAGGGTATAATGC-3′HN RP Partial 5′-CTCATGATAACTGTGGACTC-3′  402 F FP Partial5′-GGTTCAGTAGGCTCTTATAC-3′ F RP Human 5′-AGAAGGCTGGGGCTCATTTG-3′  198GAPDH FP Human 5′-AGGGGCCATCCACAGTCTTC-3′ GAPDH RP

The clone obtained after cloning was amplified and cultured in an LBliquid medium for 24 hours, and a plasmid was extracted using a plasmidextraction kit (Midiprep kit) (Invitrogen, USA), and then transducedinto hATMSCs using a liposome (lipofectamine 3000, Invitrogen) in amanufacturer's method to obtain hATMSCs (HN/F-hATMSCs) into which theHN/F genes were introduced. Total RNAs were extracted from thetransduced cell line and the expression of the HN and F genes wasanalyzed by RT-PCR and quantitative RT-PCR. The hATMSCs were used as acontrol group. Partial HN and F primers were used for confirmingexpression of HN or F mRNA using RT-PCR and quantitative RT-PCR.

As the expression analysis result, as shown in FIG. 4 , theoverexpression of the HN and F genes was confirmed. Such a resultindicates that the HN and F genes are successfully overexpressed throughliposomal transfection at the same time.

In addition, as shown in Table 1, a base sequence encoding a myc proteinwas attached to a forward primer for amplifying the HN gene and a basesequence encoding histidine was attached to a forward primer foramplifying the F gene. Thus, the expression of the HN and F proteins wasanalyzed in the HN/F-hATMSCs using antibodies against the myc proteinand the histidine (Santcruz Biotechnology, USA) by a confocal lasermicroscope (Nicon, Japan).

As a result of image analysis, as shown in FIG. 5 , it was confirmedthat the HN and F proteins were expressed on a cell surface ofHN/F-hATMSCs. These results indicate that the human adipose-derivedmesenchymal stem cells overexpressing the HN and F proteins aresuccessfully prepared.

Example 3. Evaluation of Expression of Stem Cell Markers in HN/F-hATMSCs

The expression for adipose-derived mesenchymal stem cell markers inHN/F-hATMSCs was analyzed using antibodies (Santcruz Biotechnology, USA)against positive markers CD29, CD44 and CD90 and negative markers CD34,CD45 and HLA-DR of the adipose-derived mesenchymal stem cells by a flowcytometry. The hATMSCs were used as a control group. As a result ofexpression analysis, as shown in FIG. 6 , it was confirmed that therewas no difference in expression between the positive markers CD29, CD44and CD90 and the negative markers CD34, CD45 and HLA-DR as compared withthe control group. These immunophenotyping results indicate that thehATMSCs introduced with the HN and F genes maintain properties of thestem cells.

Example 4. Evaluation of Cell Fusion Ability of HN/F-hATMSCs and NSC34Motorneuron Cell Line

In order to evaluate the cell fusion ability of the HN/F-hATMSCsprepared in Example 2, a fusion assay of the HN/F-hATMSCs marked with agreen dye and a NSC34 motoneuron cell line marked with a cell trackerCM-DiI was performed. Two types of cells were mixed in a 1.5 ml testtube at a cell number ratio of 1:1, reacted at 4° C. for 5 minutes, andthen reacted in a 37° C. cell incubator for 15 minutes. The cells weretransferred to a 6-well plate containing a DMEM culture medium (addedwith 10% FBS) added with antibiotics and then cultured at 37° C. under5% CO₂ supply for 16 hours. The cells were collected and a cell fusionrate of the HN/F-hATMSCs and the NSC34 motoneuron cell line was analyzedusing a flow cytometry. The hATMSCs were used as a control group. As aresult, as shown in FIG. 7 , it was confirmed that the cell fusion rateof the HN/F-hATMSCs and the NSC34 motoneuron cell line was increased by3.5 times or more compared to the control group. Such a result indicatesthat the cell fusion rate of the HN/F-hATMSCs and the NSC34 motoneuroncell line is increased.

In addition, an image of fused cells of the HN/F-hATMSCs showing greenfluorescence and the NSC34 motorneuron cell line showing redfluorescence may be confirmed by an image analysis through a confocallaser microscope, as shown in FIG. 7 .

Example 5. Expression Analysis of Marker of hATMSCs and Marker ofMotorneuron Cells in Fused Cells of HN/F-hATMSCs and NSC34 MotorneuronCell Line

For the fused cells of Example 4, the expression of adipose-derivedmesenchymal stem cell markers ChAT and CD105 was analyzed by a confocallaser microscope using antibodies (Santcruz Biotechnology, USA) againstcholine acetyltransferase (ChAT) as an motoneuron cell marker and anadipose-derived mesenchymal stem cell marker CD105. As a result, asshown in FIG. 8 , the expression of the motoneuron cell marker ChAT andthe adipose-derived mesenchymal stem cell marker CD105 was confirmed inthe fused cells of the HN/F-hATMSCs and the NSC34 motorneuron cell line.

These results confirm that the HN/F-hATMSCs and the NSC34 motorneuroncell line were fused, and indicate that the fused cells express all ofthe marker proteins expressed in the adipose-derived mesenchymal stemcells and the motorneuron cell line.

Example 6. Proteomics Analysis of Dying NSC34 Motorneuron Cell Line andFused Cells of HN/F-hATMSCs and NSC34 Motorneuron Cell Line

In order to find a cell damage repair mechanism through cell fusion ofdying motorneuron cells and HN/F-hATMSCs, a proteomics analysis for theannexin V-positive NSC34 motorneuron cell line in Example 2 and thefused cells in Example 4 was performed using ESI-LTQ-Orbitrap (TermoFiher) and nanoHPLC (RSLC, Dionex), and a heatmap analysis was performedusing MeV software. The NSC34 motorneuron cell line was used as acontrol group. As the analysis result, as shown in FIGS. 9 and 10 , itwas confirmed that five genes DDB1, HMGB1, MSH2, NONO and PCNA relatedwith cell repair were decreased in the dying NSC34 motorneuron cells,but increased in the fused cells of the HN/F-hATMSCs and the NSC34 motorneuron cell line, and thus, the five genes were selected as cell damagerepair target genes.

In addition, a change in expression of DDB1, HMGB1 and MSH2 among thefive genes DDB1, HMGB1, MSH2, NONO and PCNA related with the cell repairin the annexin V-positive NSC34 motorneuron cell line of Example 2 andthe fused cells of Example 4 was confirmed. In summary, the expressionof DDB1, HMGB1 and MSH2 was analyzed through RT-PCR and quantitativeRT-PCR in the annexin V-positive NSC34 motorneuron cell line of Example2 and the fused cells of Example 4. The NSC34 motorneuron cell line wasused as a control group. As shown in FIG. 11 , the expression of DDB1,HMGB1, and MSH2 mRNAs was decreased in the dying NSC34 motorneuron cellline, but was significantly increased in the fused cells of theHN/F-hATMSCs and the NSC34 motorneuron cell line.

These results indicate that in the process in which the hATMSCsoverexpressing the HN and F proteins are fused with the dying NSC34motorneuron cell line to restore the cell damage, the DDB1, HMGB1, andMSH2 are involved.

Example 7. Evaluation of Cell Fusion Ability of N2A Neuroblastoma CellLine and HeLa Cell Line HN/F-HeLa Overexpressing HemagglutininNeuraminidase (HN)/Fusion (F) Proteins

In order to find whether a cell fusion ability is shown even in ageneral cell line overexpressing HN and F proteins, a HeLa cell lineHN/F-HeLa overexpressing the HN and F proteins was prepared. In summary,a pcDNA3.1 expression vector inserted with GFP-HN was cloned using aGFP-pcDNA3.1 expression vector and the HN protein gene clone obtained inExample 2. In addition, a pcDNA3.1 expression vector inserted with aRFP-F protein was cloned using a RFP-pcDNA3.1 expression vector and theF protein gene clone obtained in Example 2. Thereafter, a liposome(lipofectamine 3000, Invitrogen) was transduced into HeLa cellsaccording to a manufacturer's method to obtain HeLa (HN/F-HeLa)introduced with HN/F genes. Total RNAs were extracted from thetransduced cell line and the expression of the HN and F genes wasanalyzed by RT-PCR and quantitative RT-PCR. The HeLa cell line was usedas a control group. As the expression analysis result, as shown in FIG.12 , the overexpression of the HN and F genes was confirmed.

In order to evaluate the cell fusion ability of the HN/F-HeLa preparedabove, the HN/F-HeLa and the N2A neuroblastoma cell line marked with theDAPI were subjected to cell fusion in the same manner as the methoddescribed in Example 4, and then the cells were collected, and the cellfusion rate of the HN/F-HeLa and the N2A neuroblastoma cell line wasanalyzed by a flow cytometry and a confocal laser microscope. As acontrol group, the N2A neuroblastoma cell line transduced with GFP and aHeLa cell line transduced with mCherry were subjected to cell fusion inthe same manner as the method described in Example 4, and the cells werecollected, and the cell fusion rate was compared and analyzed by aconfocal laser microscope. As the analysis result, as shown in FIG. 13 ,it was confirmed that the cell fusion occurred in the HN/F-HeLa and theN2A neuroblastoma cell line and the cell fusion rate of the HN/F-HeLaand the N2A neuroblastoma cell line was increased by 7 times or morecompared to the control group.

Such a result indicates that the cell fusion occurs by the HN and Fproteins regardless of the cells.

Example 8. Analysis of Gene Expression Related with Cell Repair in FusedCells of N2A Neuroblastoma Cell Line and HeLa Cell Line

In order to confirm a cell damage repair mechanism during cell fusion byHN and F proteins, as shown in FIG. 14 , it was confirmed that a bindingmotif of a TAR DNA-binding protein 43 (TDP-43) as a transcriptionalfactor (TF) was present in a promoter region of DDB1 by screening thepromoter region of a gene DDB1 related with cell repair selected inExample 6.

In order to confirm that the expression of DDB1 increases by increasingTDP-43 during cell fusion by the HN and F proteins, as shown in FIG. 15, primers specifically binding to a mouse DDB1 (mDDB1) were prepared andtotal DNAs were extracted from the N2A neuroblastoma cell line and theHeLa cell line using the primers, and then the mouse-specific DDB1 genewas confirmed by PCR. In order to confirm that the expression of theDDB1 is regulated by the TDP-43, the TDP-43 was overexpressed in the N2Aneuroblastoma cell line and then RNAs were extracted and analyzed usingquantitative RT-PCR. Thereafter, the cell fusion was performed betweenthe N2A neuroblastoma cell line and the HeLa cell line transduced withGFP and between the N2A neuroblastoma cell line and the HeLa cell linetransduced with GFP-TDP-43 using a Sendai virus (HVJ) envelope cellfusion kit (GenomONE™-CF EX Sendai virus (HVJ) Envelope Cell Fusion Kit,COSMO BIO, Japan) according to the manufacturer's method, respectively,and then a ChIP chromatin immunoprecipitation (ChIP) assay (Millipore)was performed using a fusion GFP antibody (Rockland). As a result, asshown in FIG. 16 , it was confirmed that mDDB1 was increased accordingto the fusion time in the fused cell line of the N2A neuroblastoma cellline and the HeLa cell line transduced with GFP-TDP-43, and theoccupancy of TDP-43 in the mDDB1 promoter region was increased by 4times or more, and the expression of DDB1 was regulated by increasingTDP-43.

Further, in order to confirm whether the TDP-43 is translocated into anucleus of a recipient cell during cell fusion by the HN and F proteins,the N2A neuroblastoma cell line marked with CM-DiI and the HeLa cellline transduced with GFP-TDP-43 were cell-fused in the same manner asthe method described above and then translocation of TDP-43 was analyzedby a confocal laser microscope after DAPI staining. In addition, the N2Aneuroblastoma cell line was stained using NucBlue (ThermoFisher) andcell-fused with the HeLa cell line transduced with GFP-TDP-43 in thesame manner as the method described above and then analyzed by aconfocal laser microscope after performing immunostaining using a humannuclei antibody (abcam). As the analysis result, as shown in FIG. 17 ,it was confirmed that the TDP-43 in the HeLa cell line transduced withGFP-TDP-43 is translocated into the nucleus of the N2A neuroblastomacell line.

Such a result indicates that an increase in expression of the gene DDB1related with cell repair during the cell fusion by the HN and F proteinsis regulated by binding to the promoter region of mDDB1 after TDP-43 ofa donor cell is translocated into the nucleus of the recipient cell.

Example 9. Analysis of Therapeutic Effect Using HN/F-hATMSCs in CellDamage-Related Disease Model

In order to confirm the therapeutic effect using HN/F-hATMSCs in thecell damage-related disease, G93A SOD1 Tg mice (80 days after birth) asan amyotrophic lateral sclerosis (ALS) disease model were received froma Jackson laboratory. Thereafter, the G93A SOD1 Tg mice were dividedinto a control group (Tg-saline), a Tg-MSC group, and a Tg-fusogenic MSCgroup, and as shown in FIG. 18 , a saline was injected in the controlgroup, hATMSCs were injected in the Tg-MSC group, and the HN/F-hATMSCsprepared in Example 2 were injected in the Tg-fusogenic MSC group withthe cell number of 0.5 to 2×10⁶, respectively, and then a rota rod testwas performed. As a result, as shown in FIG. 19 , it was confirmed thatin the Tg-fusogenic MSC group injected with the HN/F-hATMSCs, mobilitywas improved by the fusion of the HN/F-hATMSCs and the neuron cells bythe HN/F proteins and the neuron cell damage repair.

These results indicate that as shown in FIG. 21 , in the celldamage-related disease, the overexpressed cells of the HN/F proteins arefused with the damaged cells to translocate a transcription regulator inthe overexpressed cells of the HN/F proteins into the nucleus of thedamaged cells and regulate the expression of the gene involved in thecell repair in the damaged cells by the translocated transcriptionregulator, thereby restoring the damaged cells and treating the celldamage-related diseases.

Further, in order to confirm the therapeutic effect using theHN/F-hATMSCs in the damage-related diseases, mdx mice (2 to 4 weeksafter birth) as a Duchenne muscular dystrophy (DMD) disease model werereceived from a Jackson laboratory. Thereafter, the mdx mice weredivided into a control group (saline), a MSC group, and a fMSC group,and a saline was injected in the control group, hATMSCs marked with agreen dye was injected in the MSC group, and HN/F-hATMSCs marked with agreen dye was injected in the fMSC group with the cell number of 0.5 to2×10⁶ by using an intra-spinal cord injection, respectively, andskeletal muscles were isolated after 1 week. Survival rates of thehATMSCs and the fMSCs in the extracted skeletal muscles were analyzed bya confocal laser microscope using a green dye and a hNuclei antibody(abcam). Further, after 15 weeks of injection, the skeletal muscles wereisolated and immunohistochemistry (IHC) was performed using a dystrophinantibody (abcam). As a result, as shown in FIG. 20 , in the MSC groupand the fMSC group, the survival rates of the hATMSCs and theHN/F-hATMSCs were confirmed by a green dye, respectively, and in thefMSC group, it was confirmed that the expression of a human normaldystrophin protein was significantly increased after 15 weeks ofinjection.

These results indicate that the human normal dystrophin protein may beexpressed in the muscle of the fused mdx mice by the expression of thehuman normal dystrophyin gene by the cell fusion and thus, the cellfusion may be used as a delivery tool of gene therapy.

What is claimed is:
 1. A method of fusing stem cells or progenitor cellsto damaged nerve cells, comprising: administering stem cells orprogenitor cells to the damaged nerve cells of a subject suffering fromneurodegenerative diseases or neurological diseases, wherein the stemcells or progenitor cells are transformed with a vector including genesencoding hemagglutinin neuraminidase (HN) and fusion (F) proteins, andthe stem cells or progenitor cells transformed with said vector expressHN and F proteins and increase cell fusion with the damaged nerve cellscompared to stem cells or progenitor cells not transformed with saidvector.
 2. The method of claim 1, wherein the HN and F proteins arederived from sendai virus, human immunodeficiency virus 1, influenzavirus or vesicular stomatitis virus.
 3. The method of claim 1, whereinthe vector is any one selected from the group consisting of a linear DNAplasmid DNA, a non-viral recombinant vector, a viral recombinant vector,and an inducible gene expression vector system.
 4. The method of claim3, wherein the viral recombinant vector is any one selected from thegroup consisting of retroviruses, adenoviruses, adeno-associatedviruses, helper-dependent adenoviruses, herpes simplex viruses,lentivirus vectors, and vaccinia viruses.
 5. The method of claim 1,wherein the neurodegenerative diseases or the neurological diseases areselected from the group consisting of Alzheimer's disease (AD),dementia, multi-infarct dementia (MID), frontotemporal dementia,dementia with Lewy bodies, mild cognitive impairment, corticobasaldegeneration, Parkinson's disease (PD), multiple system atrophy (MSA),Huntington's disease, spinal muscular atrophy, spinal bulbar muscularatrophy, progressive supranuclear palsy (PSP), metabolic brain disease,depression, epilepsy, dentatorubropallidoluysian atrophy (DRPLA),spinocerebellar ataxia, amyotrophic lateral sclerosis (ALS), multiplesclerosis, primary lateral sclerosis, progressive bulbar palsy,glaucoma, stroke, brain ischemia, post-encephalitic parkinsonism,Tourette's syndrome, restless legs syndrome, and attention deficitdisorders with hyperactivity.
 6. The method of claim 1, wherein the stemcells or progenitor cells are autologous, allogenic, or xenogenic cells.7. The method of claim 1, wherein the stem cells are embryonic stemcells, adult stem cells, or induced pluripotent stem cells.